Gutiérrez-Rivera et al. Appendix A. Supplementary data

MATERIALS AND METHODS

Processing of human skin biopsies. Human skin samples were obtained from patients undergoing plastic or circumcision surgery (Table S1). All biopsies were obtained following the relevant legal and ethical guidelines. Skin biopsies were stored in RPMI-1640 medium (Sigma-Aldrich) supplemented with Penicillin-Streptomycin (P/S, 2X) and processed at a maximum 4 hours postsurgery. To obtain cell suspensions from whole skin samples, the tissue was first washed with 70% ethanol and then with PBS. After removing the fat, the skin was cut into small pieces and incubated overnight in Collagenase solution (2 mg/ml, Sigma-Aldrich) at 37ºC under agitation. Collagenase fraction was collected and the remaining skin pieces were incubated twice with 0.25% Trypsin-0.02% EDTA solution (T/E, Sigma-Aldrich) at 37ºC for 30-40 minutes. Collagenase and/or T/E fractions were neutralized with standard culture medium, filtered through a 40-µm cell strainer (BD Biosciences), centrifuged at 1,500 rpm and resuspended.

FACS analysis of cell suspensions. To study CD34 expression, cells were incubated with anti-CD34-PE antibody (BD Biosciences, clone 8G12), after addition of normal mouse serum (NMS) 1:10 (Sigma-Aldrich), for 20-30 minutes at 4ºC. Cells were then washed and analysed in a FACSCalibur equipped with Cell Quest software (BD Biosciences). At least 100,000 events were analysed. For phenotypic analysis, cells were incubated with anti-CD45-APC (BD Biosciences,clone 30-F11), anti-CD31-FITC (BD Biosciences, clone WM59), anti-vimentin-Cy3 (Sigma- Aldrich, clone V9) and anti-vimentin (Sigma-Aldrich, clone V9) antibodies. For direct antibodies, mouse IgG1 PE, FITC and APC (BD Biosciences) and Sheep anti mouse IgG-Cy3 (Sigma-Aldrich) were used for isotypic controls. For indirect antibodies Goat F(ab’)2 anti mouse IgG (H+L)-APC (BD Biosciences) was used as secondary antibody.

Isolation of CD34 positive cells using magnetic beads. CD34 progenitor cell selection system (Dynal Biotech) was used to isolate CD34 cells. Cells were resuspended in isolation buffer (Phosphate buffered saline, PBS with 2% bovine serum albumin, BSA, Fluka), 0.6 % Tri-Na-citrate dehydrate (Sigma-Aldrich) and 100 IU/ml P/S) containing DNase (3U/10 ml, Invitrogen) and incubated for 10 min. After centrifuging, cell pellets were resuspended in 1 ml isolation buffer per 40 million cells and incubated with the anti-CD34 magnetic beads previously exposed to washing buffer (PBS with 0.1% BSA), for 30 minutes at 4ºC under gentle rotation. Then, the CD34 negative fraction was recovered using a MPC-1 Magnetic Particle Concentrator (Dynal Biotech). After the addition of DETACHaBEAD (100 µl, Dynal Biotech), cells were incubated for 15 minutes at 37ºC (or 45 minutes at RT) and after that, MPC-1 magnet was applied to elute CD34+ fraction.

Immunofluorescence on coverslips. Cells were plated at a concentration of 30,000 cells/cm2 on coverslips (Menzel-Glässer) with no feeder layer. After overnight incubation in keratinocyte medium, cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 10 minutes and permeabilized with Triton X-100 (BioRad) for 10 minutes. Immunofluorescence was performed as described [1]. Anti-CD34 (Serotec, clone 8G12) and anti-vimentin (Sigma-Aldrich, clone V9) were used as primary antibodies and Goat anti mouse IgG (H+L)-alexa 488 (Molecular probes) as a secondary. The slides were mounted with Mowiol (Fluka) and images were processed on a confocal laser-scanning microscope (Zeiss LSM 510).

Keratinocyte culture. Cell fractions obtained after magnetic bead isolation were cultured at a concentration of 30,000 cells/cm2 on a lethally irradiated feeder layer of 3T3 Swiss Albino cells (ECACC, No. 85022108) under standard keratinocyte culture conditions [2].

SUPPLEMENTARY FIGURE LEGENDS

Figure S1. Detection and phenotypic characterization of CD34+ cells in human skin by flow cytometry. (A) Forward (FSC) and side (SSC) scatter of the population of cells selected (G1) for double staining analyses of a representative skin biopsy out of 44 processed is shown. Skin cell populations were obtained by enzymatic digestion with collagenase and trypsin. (B) Double stainings were performed to analyze coexpression of CD34, CD31 and CD45. Most of the CD34+ cells resulted negative for endothelial (CD31, b2) and haematopoietic (CD45, b4) cell markers. Numbers indicate percent cells in a given quadrant. Isotypic controls are shown in panels b1, b3.

Figure S2. Detection of dermal cells in skin cell fractions by flow cytometry and immunofluorescence. Cells of dermal origin were detected by vimentin expression. (A) Expression of vimentin was analyzed by either using the direct antibody anti-vimentin-Cy3 (a1-a3) or the indirect antibody anti-vimentin and a secondary antibody anti mouse IgG1 (a4-a9). In all cases, both Unsorted and CD34- fractions showed two vimentin +/- subpopulations (a1, a3; a4, a6; respectively), while CD34+ cell fraction was mainly vimentin positive (a2, a5). Isotypic controls are shown in grey. These data were confirmed through double staining with anti-CD34 antibody (a7-a9). Numbers indicate percent cells in a given quadrant. (B) Expression of vimentin was confirmed by immunofluorescence and confocal microscopy. Vimentin positive cells were detected in all fractions (b1, b3, b5). Cells presented a cytoplasmic expression of vimentin as expected (b2, b4, b6). Scale bars: 10 mm.

SUPPLEMENTARY TABLE 1

Table S1. Percent CD34+ cells in human skin biopsies.

BIOPSY / AGE / SEX / DONOR AREA / ISOLATION
EFFICIENCY
(cells/ cm2) (X 106) / CD34+ (%) / Mean ± SD
B45 / 20 / Fa / ABDOMEN / 1.7 / 3.5 / 16.7±12.2
B47 / 28 / 1.31 / 9.8
B43 / 33 / 3.2 / 6.3
B132 / 39 / 3.55 / 42.4
B49 / 44 / Mb / 10.2 / 13.8
B89 / 44 / F / ND3 / 20.7
B115 / 49 / 2.15 / 16.1
B134 / 49 / 3.08 / 21.3
B53 / 20 / F / BREAST / 6.03 / 7.3 / 12.3±6.9
B41 / 27 / 1 / 7.2
B67 / 27 / 5.3 / 8.8
B68 / 27 / 1.9 / NDc
B38 / 28 / 9.3 / 10.8
B114 / 30 / 8 / 22.7
B145 / 33 / 1.1 / 27
B33 / 34 / 1.8 / 10.5
B32 / 36 / ND3 / 9.5
B69 / 37 / 11.2 / 18.5
B39 / 39 / 1.8 / 5.2
B46 / 45 / 1.6 / 16.3
B64 / 45 / 2.7 / 11.3
B36 / 48 / 4.9 / 4.5
B113 / 42 / F / BUTTOCK / ND3 / 23.0
B63 / 31 / M / EAR / ND3 / 11.7 / 20.7±12.8
B156 / 63 / F / ND3 / 29.8
B31 / 38 / M / EYELID / ND3 / 15.0
BP55 / 28 / M / FORESKIN / ND3 / 11.2 / 10.2±5.1
BP9 / 36 / 17.5 / 12.7
BP5 / 37 / 38.6 / 5.6
BP3 / 43 / 28.3 / 4.7
BP7 / ND / 26 / 4.8
BP11 / ND / 35.8 / 17.1
BP18 / ND / 45.4 / 15.1
B144 / 28 / F / GROIN / 1.2 / 15.0 / 19.2±6.0
B148 / 38 / 4.65 / 23.4
B88 / 46 / F / SCALP / ND3 / 16.1 / 16.5±8.4
B160 / 53 / 13
B48 / 54 / 14.8
B55 / 56 / 14.0
B154 / 56 / 33.5
B155 / 59 / 11.2
B80 / 62 / M / 6.6
B52 / 68 / 12.0
B66 / 71 / F / 26.9
MEAN ± SD / 10.0 ± 12.7 / 14.7±8.4

a F, Female ; b M, Male ; c ND, Not determined.

SUPPLEMENTARY REFERENCES

1. Gago, N, Perez-Lopez, V, Sanz-Jaka, JP, Cormenzana, P, Eizaguirre, I, Bernad, A, et al.: Age-dependent depletion of human skin-derived progenitor cells. Stem Cells 27: 1164-1172, 2009.

2. Rheinwald, JG, Green, H: Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6: 331-343, 1975.